Analysis of Aggrecanase Activity Using Neoepitope Antibodies.

Published on Jan 1, 2020in Methods of Molecular Biology
· DOI :10.1007/978-1-4939-9698-8_11
Salvatore Santamaria14
Estimated H-index: 14
(Imperial College London),
Kazuhiro Yamamoto17
Estimated H-index: 17
(University of Liverpool)
Sources
Abstract
: Aggrecan is a major matrix component of articular cartilage, and its dysregulated proteolysis is a crucial event in the pathogenesis of arthritis. Aggrecanases, members of ADAMTS family, play a pivotal role in aggrecan degradation with ADAMTS-4 and ADAMTS-5 being key enzymes. Cleavage events mediated by ADAMTSs are highly specific and very well characterized; therefore, it is possible to investigate aggrecanolysis by using antibodies reacting with the new N- and C-termini of the cleavage products (neoepitope antibodies). Here, we present a method for analyzing dynamic aggrecanolysis by Western blotting using neoepitope antibodies in combination with antibodies against total aggrecan fragments. The protocol is robust and has a broad application for investigation of aggrecanase activity in vitro and ex vivo.
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